阅读说明：本技术 一种产生基因点突变的融合蛋白及基因点突变的诱导方法 (Fusion protein for generating gene point mutation and induction method of gene point mutation ) 是由 王小林 李硕 于 2019-03-26 设计创作，主要内容包括：本发明涉及一种产生基因点突变的融合蛋白及基因点突变的诱导方法。本发明找到了多种新的胞核嘧啶核苷脱氨酶,通过与nCas9/dCas9为代表的突变型核酸酶组合,得到的新融合蛋白能对位于前间隔序列1-16位的胞嘧啶(前间区序列邻近基序(PAM)的NGG序列定为21-23位)实现有效的C-T碱基突变,且不同的胞核嘧啶核苷脱氨酶为基础的融合蛋白,其可突变范围各有差异。本发明可实现更广范围、更精细的C-T单碱基替换,拓宽单碱基编辑工具的应用。(The invention relates to a fusion protein for generating gene point mutation and an induction method of the gene point mutation. The invention finds a plurality of new cytosine nucleoside deaminases, and the new fusion protein obtained by combining the cytosine deaminases with mutant nucleases represented by nCas9/dCas9 can realize effective C-T base mutation on the cytosine positioned at the 1-16 position of a pre-spacer sequence (the NGG sequence of a motif (PAM) adjacent to the pre-spacer sequence is positioned at the 21-23 position), and the fusion proteins based on different cytosine nucleoside deaminases have different mutation ranges. The invention can realize wider and more precise C-T single base substitution and broaden the application of single base editing tools.)

1. A fusion protein comprising a cytidine nucleoside deaminase that is identical to or identical to an amino acid sequence as set forth in any one of SEQ ID Nos. 1-13 and that retains cytidine deamination activity, and a nuclease.

2. The fusion protein of claim 1, wherein the nuclease is a Cas enzyme having no cleavage activity or only single strand cleavage activity.

3. The fusion protein of claim 2, wherein the nuclease is selected from the group consisting of: spCas9 or various mutants thereof, SaCas9 or various mutants thereof, Cpf1 or various mutants thereof.

4. The fusion protein of claim 3, wherein the nuclease is selected from the group consisting of nspCas9, nSaCas9, nLbCpf1, nAsCpf1, dspCas9, dSaCas9, dLbCpf1, and dAsCpf 1.

5. The fusion protein of claim 1, further comprising one or more of the following sequences: linkers, nuclear localization sequences, and amino acid residues or amino acid sequences introduced for the purpose of constructing fusion proteins, promoting expression of recombinant proteins, obtaining recombinant proteins that are automatically secreted outside host cells, or facilitating purification of recombinant proteins.

6. A polynucleotide sequence selected from the group consisting of:

(1) a polynucleotide sequence encoding the fusion protein of any one of claims 1-5; and

(2) the complement of the sequence of (1).

7. A nucleic acid construct comprising the polynucleotide sequence of claim 6.

8. The nucleic acid construct of claim 7, wherein the nucleic acid construct has a5 'to 3' structure of formula I:

P1-X1-L1-X2(I)；

wherein P1 is a first promoter sequence;

x1 is the coding sequence of cytidine deaminase;

l1 is nothing or a linking sequence;

x2 is a coding sequence for a nuclease that is a Cas enzyme with no or single strand cleavage activity;

and, each "-" is independently a bond or a nucleotide linking sequence;

or

P1 is a first promoter sequence;

x1 is a coding sequence for a nuclease that is a Cas enzyme with no or single strand cleavage activity;

l1 is nothing or a linking sequence;

x2 is the coding sequence of cytidine deaminase;

and, each "-" is independently a bond or a nucleotide connecting sequence.

9. A host cell containing or expressing the fusion protein of any one of claims 1 to 5, or containing the polynucleotide sequence of claim 6 or the nucleic acid construct of claim 7.

10. A method for inducing a point mutation in a gene, the method comprising the step of expressing or transfecting the fusion protein of any one of claims 1 to 5 and a sgRNA in a cell, wherein the sgRNA comprises a target binding region capable of specifically binding to a nucleic acid sequence to be mutated and a nuclease recognition region capable of being recognized and bound by a nuclease in the fusion protein.

Technical Field

The invention relates to the field of molecular biological gene editing, in particular to a fusion protein for generating gene point mutation and an induction method of the gene point mutation.

Background

In recent years, with the rapid development of genetic engineering technology, the CRISPR (Clustered regularly amplified polymorphic short palindromic repeats) technology has become a research hotspot in the scientific community, and is widely applied to various fields such as in vivo and in vitro genome editing, construction of transgenic animals, gene therapy and the like.

The CRISPR-Cas9 system widely applied to genome editing at present belongs to a II-type CRISPR-Cas system, and the action mechanism of the CRISPR-Cas system can be divided into three steps: the first step is recognition of foreign DNA by Cas protein, after which the Cas protein will selectively cleave foreign DNA (called protospacer) of 30-50bp in length and insert it into CRISPR site of prokaryote (host). The standard for Cas protein selection of prototype spacer sequences is nearby PAM sequences, i.e., only foreign DNA near PAM sequences can be recognized by Cas, cleaved, and inserted into CIRSPR sites. The second step is the transcription of the CRISPR site, eventually forming two short-chain crRNAs (CRISPR-derived RNAs) and tracerRNA (trans-acting crRNA). Wherein the crRNAs comprise a sequence complementary to the exogenous DNA, which is the basis for the CRISPR-Cas system to recognize and clear the exogenous DNA. The third step is to remove the invading foreign DNA. This process requires the combined action of Cas9, crRNA and tracerRNA, which target exogenous double-stranded DNA, the specificity of targeting being determined by the exogenous DNA complement contained in the crRNA: DNA double strand break nicks (DSBs) are formed only if the foreign double stranded DNA contains a sequence complementary to the crRNA and has a PAM site to be cleaved by the complex formed by Cas9, crRNA and tracerRNA.

From the CRISPR-Cas9 mechanism of action described above we can see that the CRISPR-Cas9 system cleaves exogenous DNA dependent on Cas9, crRNA and tracerRNA. Specific crRNA can be constructed by searching a prototype spacer sequence in the genome of the eukaryote, and then by using Cas9 and tracerRNA as auxiliary materials, the genome of the eukaryote can be directionally cut. However, researchers fuse crRNA and tracerRNA to construct a single sgRNA (single-stranded RNA), so that the whole system can perform genome-directed cleavage only by Cas9 and the sgRNA. Cas9 and sgrnas can introduce DNA Double Strand Breaks (DSBs) in the genome to initiate the gene editing process.

The basic principle of traditional genomic point mutation induction and repair is to use double-strand gaps (DSBs) induced or induced in the target site region, which activate the DNA repair mechanism in the cell to perform genome modification, such as Non-Homologous end join (NHEJ) or Homologous Recombination (HR). Homologous recombination typically occurs by requiring exogenous template DNA to repair mutations near DSBs or to introduce point mutations near DSBs.

The probability of spontaneous DSB generation in mammalian cells is less than about 1/10⁴If the DSBs are induced by adopting nucleases such as spCas9, SaCas9 and the like through a genetic engineering method, the efficiency can be improved to more than 10 percent, and the site specificity is realized, so that the gene repair process of the endogenous gene target site is facilitated to be smoothly carried out in the next step. The introduction of DSBs during point mutation induction and repair favors the development of NHEJ, and strategies to inhibit NHEJ are used to enhance the efficiency of HR due to the competitive relationship between NHEJ and HR. Nevertheless, the efficiency of HR is still not high.

In nucleases such as spCas9 and SaCas9, specific point mutations (such as D10A mutation and N863A mutation in spCas9) inhibit the ability to cleave double-stranded DNA to form DSBs, so that the nuclease cleaves only one strand of double-stranded DNA (single point mutations such as D10A or N863A in spCas9, called nickase nCas9, wherein D10A mutant Cas9 cleaves a single-stranded DNA complementary to sgRNA, and N863A mutant Cas9 cleaves a non-complementary strand) or does not cleave double-stranded DNA (multiple point mutations, such as introduction of both D10A and N863A point mutations in spCas9, called inactivated body Cas9(dCas 9)); however, the mutated nuclease can still recognize and bind to a specific DNA sequence under the guidance of the sgRNA.

Combining a specific cytidine deaminase (cytosine deaminase) with a mutant nuclease (such as spCas9 carrying D10A or/and N863A mutation), under the guidance of sgRNA, the obtained fusion protein can directly induce the mutation of cytosine (C) to uracil (U) at a specific position of a pre-spacer sequence (protospacer) under the condition of not inducing DSBs, and the uracil (U) can be identified and replaced by thymine (T) in a DNA sequence, so that the single-base mutation of C-T is finally realized. An important DNA repair enzyme, uracil glycosylase (uracil glycosylase), exists in cells, and can specifically recognize and repair uracil residues in a single strand or a double strand of DNA, so that the C-T base substitution is inhibited. To increase the efficiency of single base editing, Uracil Glycosylase Inhibitor (UGI) is added to or co-expressed with the fusion protein. The cytosine nucleoside deaminases currently used for single base editing are rat cytidine deaminase APOBEC1, human activation-induced cytidine deaminase (AID), human cytidine deaminase APOBEC3A, and lamprey cytidine deaminase PmCDA 1. The fusion protein obtained by combining with different mutant nucleases (such as spCas9 and various mutants; SaCas9 and various mutants; Cpf1) can realize C-T base mutation in a specific region, wherein the mutation range depends on two aspects, namely, a pre-spacer sequence adjacent to a PAM site which can be recognized by the nucleases and an active region of the cytosine nucleoside deaminase. It is known that a fusion protein of PmCDA1 of rat APOBEC1 and lamprey in combination with spCas9 nickase mainly edits cytosine at positions 4 to 8 of a pre-spacer (the NGG sequence of a pro-spacer adjacent motif (PAM) is defined as positions 21 to 23), and has a limited ability to edit cytosine at other positions. Further, patent document CN109021111A, publication No. 2018.12.18, discloses a gene base editor comprising two fragments, the first fragment comprising apolipoprotein B human cytosine deaminase 3A (human APOBEC3A, hA3A) and the second fragment comprising CRISPR/Cas system-associated protein. This gene base editor can achieve highly accurate and highly efficient targeted base editing even in the background of GpC dinucleotides, and can also efficiently edit methylated cytosine (methylated C). Patent documents CN107522787A, 2017.06.15 disclose a fusion protein that generates point mutations in cells, which contains or is formed by Cas enzymes with deleted cytosine deaminase and nuclease activities and retained helicase activities, and the preparation and use thereof. The invention can realize site-directed mutagenesis and obtain high mutagenesis efficiency and various mutagenesis combinations in a specific gene region.

However, the insufficient editing capability of the current single-base editing tool greatly limits the application of the single-base editing tool.

Disclosure of Invention

The invention aims to provide a novel single-base editing tool aiming at the defects in the prior art.

In a first aspect, the invention provides a fusion protein comprising a cytidine deaminase that is identical to, or has the same identity as, an amino acid sequence as set forth in any one of SEQ ID NOs 1-13 and that still retains cytidine deaminase activity, and a nuclease.

As a preferred example, the nuclease is a Cas enzyme having no cleavage activity or only single strand cleavage activity.

As another preferred example, the nuclease is selected from: spCas9 or various mutants thereof, SaCas9 or various mutants thereof, Cpf1 or various mutants thereof.

As another preferred example, the nuclease is selected from nspCas9, nSaCas9, nLbCpf1, nAsCpf1, dspCas9, dSaCas9, dlbcf 1, dAsCpf 1.

As another preferred example, the fusion protein further comprises one or more of the following sequences: linkers, nuclear localization sequences, and amino acid residues or amino acid sequences introduced for the purpose of constructing fusion proteins, promoting expression of recombinant proteins, obtaining recombinant proteins that are automatically secreted outside host cells, or facilitating purification of recombinant proteins.

In a second aspect, the present invention provides a polynucleotide sequence selected from:

(1) a polynucleotide sequence encoding any one of the fusion proteins; and

(2) the complement of the sequence of (1).

In a third aspect, the invention provides a nucleic acid construct comprising the polynucleotide sequence.

As a preferred example, the nucleic acid construct is an expression vector for expressing the fusion protein in a host cell.

As another preferred example, the nucleic acid construct has a5 'to 3' structure of formula I:

P1-X1-L1-X2(I)；

wherein P1 is a first promoter sequence;

x1 is the coding sequence of cytidine deaminase;

l1 is nothing or a linking sequence;

x2 is a coding sequence for a nuclease that is a Cas enzyme with no or single strand cleavage activity;

and, each "-" is independently a bond or a nucleotide linking sequence;

or

P1 is a first promoter sequence;

x1 is a coding sequence for a nuclease that is a Cas enzyme with no or single strand cleavage activity;

l1 is nothing or a linking sequence;

x2 is the coding sequence of cytidine deaminase;

and, each "-" is independently a bond or a nucleotide connecting sequence.

In a fourth aspect, the invention provides a host cell comprising or expressing any one of the fusion proteins, or comprising the polynucleotide sequence or the nucleic acid construct.

In a fifth aspect, the present invention provides a method for inducing a gene point mutation, the method comprising the step of expressing or transfecting any one of the fusion protein and a sgRNA in a cell, wherein the sgRNA comprises a target binding region capable of specifically binding to a nucleic acid sequence to be mutated and a nuclease recognition region capable of being recognized and bound by a nuclease in the fusion protein.

As a preferred example, the method determines the design of a cytidine deaminase, nuclease, or sgRNA with reference to the information in table 5 of the present invention.

The invention has the advantages that:

1. the invention finds a plurality of new cytosine nucleoside deaminases, and the new fusion protein obtained by combining the cytosine deaminases with mutant nucleases represented by nCas9/dCas9 can realize effective C-T base mutation on the cytosine positioned at the 1-16 position of a pre-spacer sequence (the NGG sequence of a motif (PAM) adjacent to the pre-spacer sequence is positioned at the 21-23 position), and the fusion proteins based on different cytosine nucleoside deaminases have different mutation ranges. Based on this, a novel gene editing composition is provided, which can realize a wider range and more precise C-T single base substitution.

2. The invention also discovers that the combination mode of the cytidine deaminase and mutant nuclease represented by nCas9/dCas9 has important influence on the C-T base mutation capability of the fusion protein. When cytidine deaminase is fused at the amino terminus (N-terminus) of a mutant nuclease represented by nCas9/dCas9, the resulting fusion protein generally has a higher single-base editing activity, and a broader range of C-T base mutations. When the cytidine deaminase is fused at the carboxyl terminus (C-terminus) of a mutant nuclease represented by nCas9/dCas9, the single-base editing activity of the fusion protein is significantly reduced, but the single-base editing activity of a part of the fusion protein is still not significantly changed, but the C-T base mutation range is significantly reduced. Namely, the invention proves that different fusion methods can change the action range of C-T single base substitution.

In general, the present invention broadens the application of single base editing tools.

Drawings

FIG. 1 shows a cytidine deaminase and its corresponding single base editing system.

FIG. 2 shows the information of the expression vector of the single base editing system.

Fig. 3 is the editing efficiency of the N-terminal fusion single base editing system for the four sgrnas sgA, sgB, sg18, and sg 19.

Fig. 4 is the editing efficiency of the C-terminal fusion single base editing system for the four sgrnas sgA, sgB, sg18, and sg 19.

Detailed Description

Cytosine nucleoside deaminase

Cytosine nucleoside deaminases are a class of enzymes that remove the amino group of cytosine molecules. In the present invention, the cytidine deaminase is an enzyme that has the same amino acid sequence as that shown in any one of SEQ ID Nos. 1 to 13, or has the same identity thereto, and still maintains deamination activity. For example, variants and mutants having a certain level (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%) of sequence identity, which all have cytidine deamination activity.

The cytosine nucleoside deaminase of the invention may also be further modified at some amino acid positions, such as by addition, deletion and/or substitution. Such modifications may be substitution substitutions made at one, two or three or more amino acid positions. In one embodiment, the modification is a substitution at one position. In some embodiments, such substitutions are conservative amino acid substitutions.

"conservative amino acid substitution" refers to the situation where an amino acid residue is substituted with another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been well-defined in the art, and include the families of basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-critical amino acid residues in the fusion proteins disclosed in this invention can be substituted with another amino acid residue from the same side chain family.

In the present invention, there are various strategies to introduce the cytosine nucleoside deaminase into specific mutations by recognizing specific DNA sequences, including genetically engineered I-sceI, I-aniI, FoxI, Cas9, as well as some synthetic polynucleotides, such as LNA, PNA, etc.

Cas protein

Cas protein refers to a nuclease. It includes various Cas proteins and variants thereof well known in the art, including but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7 (also referred to as Csn 7 and Csx 7), Cas7, Csy 7, Cse 7, Csc 7, Csa 7, Csn 7, Csm 7, Cmr 7, Csb 7, Csx 7, CsaX 7, Csx 36f 7, Csx 36f 7, Csx 36f 36.

One preferred Cas protein is the Cas9 protein. Cas9 enzymes may be Cas9 enzymes from different species, including but not limited to Cas9 from streptococcus pyogenes (SpCas9), Cas9 from staphylococcus aureus (SaCas9), and Cas9 from streptococcus thermophilus (St1Cas9), among others. In the present invention, the Cas9 protein is a mutated Cas9 protein, specifically, a mutated Cas9 protein having no cleavage activity or only single strand cleavage activity.

sgRNA

sgrnas typically comprise two parts: a target binding region and a Cas protein recognition region. The target binding region and the Cas protein recognition region are typically linked in a5 'to 3' orientation.

The target binding region is typically 15 to 25 bases in length, more typically 18 to 22 bases, such as 20 bases in length. The target binding region specifically binds to the template strand of DNA, thereby recruiting the fusion protein to a predetermined site. Typically, the opposite region of the sgRNA binding region on the DNA template strand is immediately adjacent to the PAM, or separated by several bases (e.g., within 10, or within 8, or within 5). Therefore, when designing sgrnas, the PAM of the enzyme is usually determined according to the Cas enzyme used, then a site that can serve as PAM is found on the non-template strand of DNA, and then a fragment 15 to 25 bases long, more usually 18 to 22 bases long, immediately downstream of the PAM site of the non-template strand (3 'to 5') or separated from the PAM site by 10 or more (e.g., within 8, within 5, etc.) is used as a sequence of a target binding region of the sgRNA.

The Cas protein recognition region of the sgRNA is determined according to the Cas protein used, as will be appreciated by those skilled in the art.

Therefore, the sequence of the target binding region of the sgRNA of the present invention is a fragment 15 to 25 bases, more typically 18 to 22 bases, immediately downstream of the DNA strand containing the PAM site recognized by the selected Cas enzyme, or separated from the PAM site by 10 or more (e.g., 8 or less, 5 or less, etc.); the Cas protein recognition region is specifically recognized by the selected Cas enzyme.

Sgrnas can be prepared using methods conventional in the art, e.g., synthesized using conventional chemical synthesis methods. The sgRNA can also be transferred into a cell via an expression vector, and the sgRNA is expressed in the cell. Expression vectors for sgrnas can be constructed using methods well known in the art.

Fusion proteins

The fusion protein provided by the invention comprises a cytosine nucleoside deaminase and a nuclease. The term "comprising" does not mean that the fusion protein includes only a nuclease and a cytidine deaminase, and it is to be understood that the fusion protein may include only a nuclease and a cytidine deaminase, or may also contain other moieties that do not interfere with the targeting of the nuclease and the function of the cytidine deaminase in the fusion protein, including but not limited to various linker sequences, nuclear localization sequences, and amino acid sequences introduced into the fusion protein as described below by gene cloning procedures, and/or for the purpose of constructing the fusion protein, promoting expression of the recombinant protein, obtaining a recombinant protein that is automatically secreted outside of the host cell, or facilitating detection and/or purification of the recombinant protein, etc.

Polynucleotide sequences, nucleic acid constructs, hosts

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The nucleotide sequence of the present invention can be obtained by PCR amplification method. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

The nucleic acid constructs of the invention comprise the coding sequences of the fusion proteins of the invention, and one or more regulatory sequences operably linked to these sequences. The coding sequence of the fusion protein of the invention can be manipulated in a variety of ways to ensure expression of the protein. The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. For example, the polynucleotide sequences of the present invention may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements. The expression vector may also include a ribosome binding site for translation initiation and a transcription terminator. The polynucleotide sequences of the present invention are operably linked to a suitable promoter in an expression vector to direct mRNA synthesis via the promoter.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. Methods well known to those skilled in the art can be used to construct expression vectors containing a polynucleotide sequence of the invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The vector of the present invention may be transformed into an appropriate host cell so that it can express the fusion protein of the present invention. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; filamentous fungal cells, or higher eukaryotic cells, such as mammalian cells. The host cell may also be a plant cell. Representative examples of host cells are: e.coli; streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, filamentous fungi; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells. In addition to cells for expressing fusion proteins, other cells containing the polynucleotide sequences or vectors described herein and sgrnas or expression vectors thereof, e.g., cells for producing point muteins, are also within the scope of the host cells described herein.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

After transformation of the host cell, the obtained transformant may be cultured by a conventional method to allow expression of the fusion protein of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The recombinant fusion proteins of the present invention can be isolated and purified using various isolation methods known in the art. Such methods are well known to those skilled in the art and include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Composition and kit

The fusion protein of the invention, its coding sequence or expression vector, and/or sgRNA, its coding sequence or expression vector may also be provided in the form of a composition. For example, a composition may contain a fusion protein of the invention and a sgRNA or an expression vector for a sgRNA, or may contain an expression vector for a fusion protein of the invention and a sgRNA or an expression vector for a sgRNA. In the composition, the fusion protein or its expression vector, or the sgRNA or its expression vector may be provided in the form of a mixture, or may be packaged separately. The composition may be in the form of a solution or a lyophilized form.

The composition may be provided in a kit. Accordingly, the present invention provides kits comprising the compositions of the present invention. Alternatively, the present invention also provides a kit containing the fusion protein of the present invention and sgRNA or an expression vector for sgRNA, or an expression vector containing the fusion protein of the present invention and sgRNA or an expression vector for sgRNA. In the kit, the fusion protein or its expression vector, or the sgRNA or its expression vector may be packaged separately, or provided in the form of a mixture. The kit may also include, for example, reagents for transferring the fusion protein or its expression vector and/or sgRNA or its expression vector into a cell, as well as instructions directing the skilled artisan to perform the transfer. Alternatively, the kit may further comprise instructions directing the skilled artisan to practice the various methods and uses described herein using the components contained in the kit. Other reagents, such as reagents for PCR, etc., are also included in the kit.

Method and use

The method for inducing a gene point mutation of the present invention includes the step of expressing or transfecting the fusion protein and sgRNA of the present invention in a cell. A specific induction method comprises the following steps: designing sgRNA according to the characteristics of a target site (sequence information around a C site to be edited), constructing an sgRNA expression vector, selecting an appropriate expression vector of fusion protein, cotransfecting the sgRNA expression vector and the expression vector into a target cell or an animal, and realizing C-T single base replacement of the target site. Another specific inducing method is as follows: the sgRNA aiming at the target can be synthesized in vitro, mRNA for expressing the fusion protein is obtained by in vitro transcription, or the fusion protein is obtained, the mRNA or the fusion protein and the sgRNA are mixed and transfected into a target cell or an animal, and the C-T single base substitution of the target site is realized.

The cell can be any cell of interest, including prokaryotic cells and eukaryotic cells, such as plant cells, animal cells, microbial cells, and the like. Particularly preferred are animal cells, e.g., mammalian cells, rodent cells, including human, equine, bovine, ovine, murine, rabbit, and the like. Microbial cells include cells from various microbial species known in the art, particularly those having medical research value, production value (e.g., production of fuels such as ethanol, production of proteins, production of fats and oils such as DHA). The cells may also be cells of various organ origins, e.g. cells from human liver, kidney, skin, etc.

The animal can be any animal, preferably a mammal, such as a human, horse, cow, sheep, mouse, rabbit, etc.

The gene that generates the mutation may be derived from a microorganism, a plant, an animal, a cell, a mammal, or a human.

The method of the present invention may be an in vitro method or an in vivo method. When performed in vivo, the fusion protein of the present invention or its expression vector and sgRNA or its expression vector can be transferred into a subject, such as corresponding tissue cells, by means well known in the art, and the functional variant of interest can be screened by observing the phenotypic change of the animal. It will be appreciated that in vivo experiments, the subject may be a variety of non-human animals, particularly a variety of non-human model organisms routinely employed in the art. The in vivo experiment should also meet the ethical requirements.

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.